Tag Archives: FLX+

800 bp Read Length For Amplicon Sequencing Is Not Science Fiction

Amplicon sequencing with Roche GS JuniorAbout a year ago my colleguage Regina reported about the new possibilities of using the MiSeq system for amplicon sequencing (16S Amplicon Experiments: Which Platform to Choose?). Now, one year later still everything is true about the advantages of amplicon sequencing using the MiSeq (e.g. lower cost/base).

The main advantage of the Roche system are the long reads that are highly valuable for some applications. By ligating appropriate sequencing adaptors we can currently deliver average read length of up to 700 bp when using the GS FLX+ pipeline. Further improvements regarding the read length can be expected with the launch of a new amplicon pipeline from Roche for the Roche GS FLX+ system (planned for summer 2013).

And beside the ultra long reads on the GS FLX+ system there are still some advantages of amplicon sequencing using the GS Junior system compared to other technologies:

+ short turnaround time (starting from 5-10 working days)

+ competitive pricing

+ moderate to long reads (350 – 450 bp)

+ sufficient data output for all projects with a medium size of samples (e.g. up to 24)

What is your preferred next generation sequencing technology for amplicon sequencing? Take part in our current poll.

Summary from 4th Next Generation Sequencing Congress 2012

Attending the 4th NGS Congress 2012 at London Heathrow I can give here some interesting new facts and information about latest NGS stories which are worth to be shared.

First of all let’s talk about “long read technology” – A Roche 454 talk has been given by Todd Arnold, Vice President R&D, Roche 454.  For Roche GS Junior a new software version 2.7, with  “improved well resolution results in better quality, more robust sequencing runs”  is now available.  As a matter of fact we can confirm these new data outputs while using on our own Junior platform with this update since a while.  Depending on your samples nature  a good part of all reads will be longer than 400 bp and up to 450-480 bp (still using the Titanium Chemistry). But the FLX+ technology is NOT available and also NOT planned for GS Junior – raising the question why,  no concret details or upgrade plans could be given for GS Junior at the London congress…

The real and major highlight about Roche 454 was the description of what we call now “FLX++” sequencing. A software update (2.8) being available now for all the GS FLX systems – together with  the “pimped chemsitry kits” – Roche 454 is offering real “1000bp” Sanger-like reads (as initially aimed at launch).  Some data outputs and slides were shown that demonstrate these new and longer read lengths and also higher data outputs (figure 1). All together that counts up to almost ~1Gb of sequencing data per full PPT run.

Fig 1: Todd Arnold Roche 454 Data Heathrow 2012

Being one of the early access users of the FLX++ upgrades and software version 2.8, we can in fact confirm that the new data outputs are excellent (again depending on the quality of DNA) – in fact one can reach even better results than shown by Roche at the 4th NGS congress in London Heathrow. Here is an example:

Fig 2: Eurofins MWG Operon data with Roche GS FLX++

Of course one may argue now – “that’s nothing compared to Illumina data outputs” – and you are right in terms of the pure data volumes! But the focus here is on long read applications like e.g. sequencing and de novo assembly. And for this kind of NGS application, a modal read length of 800-950 bp or above will tune the final data outputs treamendously. You won’t believe? We can share with you some nice new project data that we have delivered for a fungal de novo sequencing project (figure 2). We were able to deliver chromosome-size scaffolds of 8.3 Mb, 6.0 Mb, 4.3 Mb, 2.8 Mb, 2.4Mb, 2.1 Mb, … when using a long read FLX++ back-bone sequencing at  8x-12x only and combining this data with short read LJD sequencing on HiSeq at 2x 100 bp. The complete data set missed only about 0.5% of all genetic information, while remaining average gap lenght was about 240 bp.  We are actually very interested to learn how 2x 250 bp read length on MiSeq will further improve this excellent data results – one shot genome sequencing at it’s best.

Interested in this kind of project data? Please learn more about our fascinating de novo sequencing & assembly results at our next NGS roadshow in 2013 or send me an email for further discussion about this topic…

Focus on FLX+ ?

In the last weeks we were continuously following the hostile bid from Roche for Illumina. Now, after the shareholder meeting, Roche didn’t extend the offer and let Illumina from the hook – for the moment?
However, this could also be good news. From my point of view there is a wide range of applications that work best with the FLX technology. In order to be competitive with all other NGS technologies Roche needs to invest more in FLX+. After launching the upgrade last year they faced continuously problems with the performance of the technique as highlighted for example in an article in InSequence last week.

I think, because the NGS future of Roche is again more focussed on FLX+, they will work even harder to get the long reads up an running on every GS FLX site – maybe a FLX+ upgrade for the GS Junior might be possible soon.

Of course it also might be that they are looking for an alternative for Illumina as highlighted by Julia Karow and Monica Heger. However, all possiblities discussed in this article – a cooperation with Life (Ion Torrent), an acquisition of Oxford Nanopore technologies or the development of a brand new technique might not be as scary for the FLX technology as Illumina because all technologies are “very early in the commercialization”.

The MiSeq Will Further Challenge the Roche 454 FLX+ Technology

Currently, Roche 454 has a unique selling proposition in providing the only sequencing technology on the market delivering long reads with high accuracy at the same time. Just last year Roche 454 launched the new GS FLX+ chemistry delivering reads with a modal read length of up to 700 bp. Long reads are crucial for de novo sequencing of genomes and transcriptomes and for sequencing of amplicons.

The current version of the Illumina MiSeq enables 2x 150 bp paired-end reads and 1.5 – 2 Gbp per run, which is slightly over the output of a typical GS FLX+ run. When working with short insert libraries of 200-250 bp, both paired-end reads will overlap and finally generate one longer read of up to 250 bp.

This read length is still not in a competitive range for Roche 454, but recently Illumina announced the launch of a MiSeq instrument upgrade by the middle of the year. According to Illumina ‘s vice president of marketing, the upgraded instrument will generate 2x 250 bp paired-end reads and up to 7 Gbp of data output. When sequencing short insert libraries with the 2x 250 bp paired-end module, reads with up to 450 bp can be generated. Thus, read length comes again closer to the read length of the Roche FLX+ technology (not to mention the 10x higher data output).

We have to wait and see whether the MiSeq upgrade keeps what Illumina promises, but for me personally it is quite clear, that with the announced specifications, the MiSeq will sooner or later replace Roche 454 sequencing for certain applications. In this light it is very interesting that Roche offered a friendly take over of Illumina this week.

 

Comparison of De Novo Assemblies of the Escherichia coli Outbreak

The E. coli EAHEC outbreak inGermany has been an opportunity to compare currently available sequencing technologies with respect to the data quality.

Regarding the N50 contig size and amount of contigs/scaffolds best assembly quality so far was achieved using the long read technologies in the market, Roche’s GS Junior sequencing and Pacific Biosciences’ PacBio RS sequencing. For further comparison I am therefore going to focus on data of both long read technologies. But first, please have a look at the sequencing layouts:

Sequencing layout PacBio RS (Source: Pacific Biosciences >):

First library: Standard sequencing library (200-fold coverage)
Second library: Circular consensus sequencing library (35-fold coverage)
Sequencing: 56 SMRT cells


Sequencing layout Roche GS Junior
(Source: UK Health Protection Agency HPA >):

First library: Shotgun library
Second library: Long paired end library (LPE, 8 kbp insert length)
Sequencing: Three Roche GS Junior runs (25-fold coverage)


Comparison of the results
:

The de novo assembly is comprised of 33 contigs with PacBio RS sequencing and 13 scaffolds with Roche GS Junior. The N50 contig size of the PacBio sequencing approach is 402 kbp and the N50 scaffold size of the Roche 454 sequencing approach is 968 kbp. Both de novo assemblies > with Illumina MiSeq and Ion Torrent PGM data so far revealed higher amount of contigs and considerably shorter N50 contig sizes (95 kbp and 50 kbp, respectively).

This data once again shows that de novo sequencing strictly needs long reads. The advantageous effect of the very long reads >  of PacBio for scaffolding (on average 2900 bp and 5% longer than 5100 bp) is balanced in the other approach by sequencing of the LPE library.

Important to mention is that the PacBio assembly is generated with reads from a not yet released chemistry (planned for quarter 4). In contrast Roche 454 assembly did not contain the long FLX+ chemistry reads that will become available for GS FLX by the end of the month. According to our experience a read length of 650 – 750 bp will have some additional positive effect on number of scaffold and N50 scaffold size.

Most striking for me is that as much as 56 flow cells were needed to generate the PacBio assembly with the high consensus accuracy > of 99.998 %. The standard library was sequenced with that high coverage in order to increase the number of very long reads and the circular consensus sequencing library was employed for further correction of errors derived of still low single read accuracy.